Deep Sequencing

However, aKG is also a cofactor of histone-demethylases and it remains unclear whether BCAT1 also affects these enzymes, which regulate chromatin structure and activity.Material and methods Genome wide approaches (i.e. ChIPseq for specific histone marks, ATACseq, RNAseq, DNA methylation) are used to mechanistically dissect the connexion between BCAT1 mediated metabolic changes and alterations in the chromatin landscape of AML cell lines (HL-60, SKM1-MOLM-13) and LSCs, Results and discussions In order to understand if BCAT1 is controlling the chromatin structure, the transcription factor binding and the histone methylation status, we performed H3K27ac-H3K27me3-H3K4me1-H3K4me3 ChIPseq experiments and ATACseq on control and BCAT overexpressing cells. Despite the global DNA methylation changes observed in BCAT1 overexpressing cells,Raffel et al., Nature 2017 ChIPseq and ATACseq analyses did not show a global change in chromatin structure and modifications. Nonetheless, differences in specific loci were identified after BCAT1 overexpression suggesting a more complicated scenario and opening the road for further analyses. Conclusion This work will provide rationale for the use of metabolic enzyme inhibitors (alone or in combination with epigenetic inhibitors) as a therapeutic approach in AML and other solid BCAT1 overexpressing tumour entities.

Deep Sequencing PO-396

ABSTRACT WITHDRAWN

PO-397

DNA DAMAGE TOLERANCE IS ESSENTIAL FOR THE DNA DAMAGE RESPONSE NETWORK AND HEMATOPOIETIC STEM CELL MAINTENANCE

1

B Pilzecker*, 2OA Buoninfante, 3JY Song, 4C Pritchard, 2IJ Huijbers, 5J Vivié, 6S Philipsen, PCM Van den Berk, 2H Jacobs. 1NKI/AVL, Tumor biology and immunology, Amsterdam, The Netherlands; 2The Netherlands Cancer Institute, Division of Tumor Biology and Immunology, Amsterdam, The Netherlands; 3The Netherlands Cancer Institute, Division of Experimental Animal Pathology, Amsterdam, The Netherlands; 4The Netherlands Cancer Institute, Mouse Clinic for Cancer and Aging research MCCA Transgenic Facility, Amsterdam, The Netherlands; 5Hubrecht Institute, Single cell sequencing facility, Utrecht, The Netherlands; 6Erasmus MC, Department of Cell Biology, Rotterdam, The Netherlands 2

10.1136/esmoopen-2018-EACR25.423

Introduction Stem cell fitness dictates essential biological processes like tissue homeostasis and ageing. The overall contribution of DNA damage tolerance (DDT) in maintaining stem cell fitness remains unknown. DDT pathways which enable replication in the presence of DNA replication impediments are facilitated by PCNAK164 ubiquitination and REV1. By intercrossing PcnaK164R mutant and Rev1 deficient mice, DDT was found to be essential for mammalian life. Material and methods We crossed mouse models with Rev1 deletion and PcnaK164R mutation. We use flow cytometry, pathology, and single cell RNA sequencing in these mouse models to determine the relevance of DDT in mice. Results and discussions By intercrossing PcnaK164R mutant and Rev1 deficient mice, DDT was found to be essential for mammalian life. A compound mutation of Rev1 and PcnaK164R rendered hematopoietic stem cells (HSCs) and their immediate precursors genetically instable, instigating a pathological A178

process where the associated HSC depletion culminated in a severe embryonic lethal anaemia. Single cell RNA-sequencing of the remaining LSK cells revealed a remarkable stressinduced plasticity of multipotent progenitors, and the existence of a novel CD24ahigh,CD93low erythroid-committed progenitor (ECP) compartment. Conclusion DDT is a key activity within the DNA damage response network, where PCNAK164 and REV1 primarily serve non-epistatic DDT pathways and are essential in maintaining HSCs. Furthermore, we reveal a novel CD24ahigh,CD93low erythroid-committed progenitor (ECP) within the LSK compartment.

PO-398

BENCHMARKINGOF AMPLICON-BASED NEXTGENERATION SEQUENCING PANELS COMBINED WITH BIOINFORMATICS SOLUTIONS FOR BRCA1 AND BRCA2 ALTERATION DETECTION

P Vilquin*, J Vendrell, M Larrieux, C Van Goethem, J Solassol. CHU Montpellier, Departement de Pathologie et Oncobiologie, Montpellier, France 10.1136/esmoopen-2018-EACR25.424

Introduction Fast and accurate detection of BRCA1 and BRCA2 germline alterations is at utmost importance in both prevention and therapeutic aims. High-throughput NGS is increasingly being applied in clinical diagnosis for genetic testing that reduces the cost and turnaround time. As several commercial multiplex amplicon-based targeted NGS kits and bioinformatics solutions are now available for BRCA testing, the aim of this study was to evaluate the best approach for the identification of single-nucleotide variants (SNVs), insertion/deletion variants (indels) and copy number variations (CNVs). Material and methods Four assays (BRCA HC, BRCA Tumour, Access Array BRCA, and Ion AmpliSeq BRCA) and two bioinformatics software programs (Sophia DDM platform and SeqNext software) were tested on a training set of 28 previously genotyped samples. The most relevant solution was then blindly tested on a larger cohort of 152 samples. Results and discussions In the training set, the BRCA Tumour and Access array BRCA panels provide the highest performance in terms of coverage uniformity. For the Ion AmpliSeq BRCA panel, exons 20 and 23 of BRCA2 showed a systematic drop in coverage and it also displayed an important issue of low-level of overlapping amplicons. Moreover, false positive variants were only reported using this assay independently of the bioinformatics analysis. Except for the Access Array BRCA, the analysis of CNVs has been successfully performed in the assays. Regardless of the enrichment kit used for library preparation, Sophia DDM outperforms SeqNext for CNV analysis. Our analysis performed on the training set suggests that, in terms of variant detection accuracy in BRCA1 and BRCA2, the BRCA Tumour and the BRCA HC panels are the most appropriate solutions. As the BRCA HC panel features lower coverage uniformity, the BRCA Tumour panel was preferred for the validation step. As for the training set, a good/excellent accuracy was achieved by the selected workflow (BRCA Tumour and Sophia DDM) for BRCA alterations detection in the independent validation set. Conclusion To our knowledge, a direct and systematic comparison of these Amplicon–based panel NGS methods has so far never been performed. Among the four solutions, that BRCA HC and BRCA Tumour panels exhibited suitable data for SNVs/indels and CNV analysis. Interestingly, the use of the ESMO Open 2018;3(Suppl 2):A1–A463

ESMO Open: first published as 10.1136/esmoopen-2018-EACR25.424 on 29 June 2018. Downloaded from http://esmoopen.bmj.com/ on 1 September 2018 by guest. Protected by copyright.

Abstracts

BRCA Tumour Kit combined with the Sophia DDM platform provides a powerful tool for reliable detection of genetic alterations in BRCA1 or BRCA2.

PO-399

GENOME WIDE HISTONE MODIFICATION PATTERNS AT THE PROMOTER REGIONS ARE DISTINCT IN LOW AND HIGH GRADE GLIOMAS

1

K Stepniak*, 1J Mieczkowski, 2A Macioszek, 1B Wojtas, 1B Gielniewski, 3T Czernicki, W Grajkowska, 5K Kotulska, 2B Wilczynski, 1B Kaminska. 1Nencki Institute of Experimental Biology, Neurobiology Center, Warsaw, Poland; 2University of Warsaw, Faculty of Mathematics- Informatics- and Mechanics, Warsaw, Poland; 3Medical University of Warsaw, Neurosurgery Department and Clinic, Warsaw, Poland; 4Children’s Memorial Health Institute, Laboratory of Oncopathology and Medical Biostructure, Warsaw, Poland; 5 Children’s Memorial Health Institute, Clinics of Neurology and Epileptology, Warsaw, Poland 4

10.1136/esmoopen-2018-EACR25.425

Introduction Growing evidence indicates global dysregulation of epigenetics in gliomas as a driving force of transcriptional changes and pathogenic mechanisms. Gliomas, the most common primary brain tumours, are clinically divided by WHO into 4 grades according to their malignancy. Slowly growing, pilocytic astrocytomas (PA, grade I) are the benign and most treatable of the gliomas, with a cure rate of over 90 percent. Glioblastomas (GBM, grade IV) are aggressive tumours with poor prognosis due to diffusive nature and resistance to current therapies. We endeavoured to provide a global view of epigenetic landscape of regulatory regions in combination with transcriptomes to identify gene regulatory networks that may contribute to tumorigenesis and tumour malignancy. Material and methods To achieve these goals, we collected glioma samples obtained as fresh surgical resections, produced single cell suspension and on each tumour sample we performed multilayer genome-wide analyses to identify open chromatin areas (Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) and histone modification patterns (Chromatin immunoprecipitation, ChIP-seq) in combination with transcriptome analyses (RNA-seq). Besides annotation of functional regulatory sites in tumours, we performed comparative analyses of glioma of different grades to identify dysregulations associated with tumour malignancy. Results and discussions We found that the global distribution of H3K4me3 ChIPseq peaks indicates that different glioma grades share similar genome-wide pattern of H3K4me3 enrichment in regions surrounding a transcription start site (TSS). However, we found a striking shift in enrichment levels of active histone marks around TSS from H3K4me3 towards H3K27ac in GBM. Conclusion In conclusion, this study is the very first identification of open chromatin and epigenetic regulatory networks in low and high grade gliomas. Our results demonstrate distortion of histone active marks in GBM and suggest that H3K27ac plays more important role in glioma malignancy then H3K4me3. Supported by SYMFONIA 2015/16/W/NZ2/00314 grant from the National Science Centre, Poland

PO-400

ARRIBA – FAST AND ACCURATE GENE FUSION DETECTION FROM RNA-SEQ DATA

S Uhrig*, M Fröhlich, B Hutter, B Brors. German Cancer Research Center DKFZ, Applied Bioinformatics, Heidelberg, Germany 10.1136/esmoopen-2018-EACR25.426

ESMO Open 2018;3(Suppl 2):A1–A463

Introduction Personalised oncology revolutionises the way how eligible therapies are selected to treat cancer patients. With the help of next-generation sequencing technology, cancer can be understood at the molecular level. This enables clinicians to match cancer patients to drugs that precisely target the driver mutations of the tumour. The foundation of a successful matchmaking is a reliable detection of somatic variants from next-generation sequencing data. Particularly the identification of more complex alterations such as gene fusions remains a challenging task. Material and methods We developed ’Arriba’, a novel algorithm that identifies gene fusions from RNA-seq data. It is based on the output of the STAR aligner and can easily be integrated into existing bioinformatics pipelines built on this software. The tool was designed specifically for the application in a high-throughput clinical workflow, where high sensitivity and short turn-around times are crucial. Results and discussions Arriba excels previous methods in terms of sensitivity without compromising precision. Its highly efficient algorithm reduces the time required for results generation from many hours to just a few minutes. Moreover, the tool comes with a rich set of features that are useful in a clinical context, including comprehensive annotation, assistance with the design of primers for Sanger validation, and automatic generation of publication-quality figures. Conclusion The identification of gene fusions from RNA-seq data used to be an error-prone and computationally demanding task. Arriba is a novel algorithm that overcomes these shortcomings. It delivers more accurate results than previous methods within a fraction of the time.

PO-401

DETECTION OF MUTATIONAL PATTERNS ASSOCIATED TO HR DEFICIENCY FROM LOW COUNTS OF MUTATIONS

1 D Gulhan*, 2P Park, 2JKJ Lee, 2G Melloni. 1Harvard Medical School, Deparment of Biomedical Informatics, Boston, USA; 2Harvard Medical School, Department of Biomedical Informatics, Boston, USA

10.1136/esmoopen-2018-EACR25.427

Introduction A common source of tumorigenesis in several cancer types such as breast and ovarian is a defect in the homologous recombination (HR) machinery. HR defect can be detected by identifying single nucleotide variants (SNVs), deletions, duplications, and other errors generated by the alternative doublestrand break repair machineries. However, current diagnostic tools for HR defect relies on copy number changes with reduced sensitivity and specificity, or on the existence of BRCA mutations. While, recent efforts to detect HR defect using mutational signature require a large number of SNVs, beyond what is typically obtained from panel sequencing which is widely used in clinics. Material and methods We developed a computational software called SigMA to identify signatures of HR defect even from low SNV counts. SigMA carries out a multivariate analysis to isolate the effect of a single biological process in the presence of multiple mutagenic processes. The multivariate analysis includes likelihood estimations, which we suggest as novel and sensitive measures, together with commonly used measures such as cosine similarity, and signature amplitudes. First, we determine the signature composition of SNVs in 720 wholegenome sequenced (WGS) breast cancer samples, with nonnegative matrix factorization (NMF). Then, a subset of A179

ESMO Open: first published as 10.1136/esmoopen-2018-EACR25.424 on 29 June 2018. Downloaded from http://esmoopen.bmj.com/ on 1 September 2018 by guest. Protected by copyright.

Abstracts